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Activation of R235A mutant orotidine 5'-monophosphate decarboxylase by the guanidinium cation: effective molarity of the cationic side chain of Arg-235.

ABSTRACT: The R235A mutation at yeast orotidine 5'-monophosphate decarboxylase (OMPDC) results in a 1300-fold increase in K(m) and a 14-fold decrease in k(cat) for decarboxylation of orotidine 5'-monophosphate, corresponding to a 5.8 kcal/mol destabilization of the transition state. There is strong activation of this mutant enzyme by added guanidinium cation (Gua(+)): 1 M Gua(+) stabilizes the transition state by ca. 3 kcal/mol. This stabilization is due to the binding of Gua(+) to the binary E(mut) x OMP complex, with a K(d) of 50 mM, to form the 9-fold more reactive ternary E(mut) x OMP x Gua(+) complex. The "effective molarity" of the cationic side chain of Arg-235 at the wild-type enzyme is calculated to be 160 M.

SUBMITTER: Barnett SA

PROVIDER: S-EPMC2819103 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Activation of R235A mutant orotidine 5'-monophosphate decarboxylase by the guanidinium cation: effective molarity of the cationic side chain of Arg-235.

Barnett Shonoi A SA Amyes Tina L TL Wood B McKay BM Gerlt John A JA Richard John P JP

Biochemistry 20100201 5

The R235A mutation at yeast orotidine 5'-monophosphate decarboxylase (OMPDC) results in a 1300-fold increase in K(m) and a 14-fold decrease in k(cat) for decarboxylation of orotidine 5'-monophosphate, corresponding to a 5.8 kcal/mol destabilization of the transition state. There is strong activation of this mutant enzyme by added guanidinium cation (Gua(+)): 1 M Gua(+) stabilizes the transition state by ca. 3 kcal/mol. This stabilization is due to the binding of Gua(+) to the binary E(mut) x OMP ...[more]

PMID: 20050635

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Project description:The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 1023-fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p Ka that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p Ka = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO2 by H+ that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

Dataset Information

Activation of R235A mutant orotidine 5'-monophosphate decarboxylase by the guanidinium cation: effective molarity of the cationic side chain of Arg-235.

Publications

Activation of R235A mutant orotidine 5'-monophosphate decarboxylase by the guanidinium cation: effective molarity of the cationic side chain of Arg-235.

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